BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions ...to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1‐related protein complexes and their roles in maintaining genomic stability and tumour suppression.
BRCA1 is a breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a high risk of breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability, through the formation of multiple different protein complexes with roles in DNA repair, checkpoint control, transcriptional regulation, apoptosis and mRNA‐splicing. This review outlines the different complexes in which BRCA1 is involved and their role in the maintenance of genomic stability and tumour suppression.
Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in ...DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.
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•BRCA1 forms a complex with mRNA splicing proteins following DNA damage•The BRCA1-mRNA splicing complex promotes the stability of genes involved in the DDR•BRCA1-dependent splicing of DDR genes maintains their protein expression•The BRCA1-mRNA splicing complex is required for DNA repair and genomic stability
BRCA1 functions to maintain genomic integrity by forming multiple protein complexes with different functions. Here, Savage et al. report the identification of a BRCA1 protein complex that promotes the mRNA splicing and expression of genes required for maintaining genomic stability.
5-fluorouracil (5-FU) is widely used in the treatment of cancer. Over the past 20 years, increased understanding of the mechanism of action of 5-FU has led to the development of strategies that ...increase its anticancer activity. Despite these advances, drug resistance remains a significant limitation to the clinical use of 5-FU. Emerging technologies, such as DNA microarray profiling, have the potential to identify novel genes that are involved in mediating resistance to 5-FU. Such target genes might prove to be therapeutically valuable as new targets for chemotherapy, or as predictive biomarkers of response to 5-FU-based chemotherapy.
Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging ...chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.
We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.
We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.
We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.
There is no method routinely used to predict response to anthracycline and cyclophosphamide-based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no ...benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.
DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided.
In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval Cl =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population.
A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide-based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.
mRNA splicing and export plays a key role in the regulation of gene expression, with recent evidence suggesting an additional layer of regulation of gene expression and cellular function through the ...selective splicing and export of genes within specific pathways. Here we describe a role for the RNA processing factors THRAP3 and BCLAF1 in the regulation of the cellular DNA damage response (DDR) pathway, a key pathway involved in the maintenance of genomic stability and the prevention of oncogenic transformation. We show that loss of THRAP3 and/or BCLAF1 leads to sensitivity to DNA damaging agents, defective DNA repair and genomic instability. Additionally, we demonstrate that this phenotype can be at least partially explained by the role of THRAP3 and BCLAF1 in the selective mRNA splicing and export of transcripts encoding key DDR proteins, including the ATM kinase. Moreover, we show that cancer associated mutations within THRAP3 result in deregulated processing of THRAP3/BCLAF1-regulated transcripts and consequently defective DNA repair. Taken together, these results suggest that THRAP3 and BCLAF1 mutant tumors may be promising targets for DNA damaging chemotherapy.
Digital image analysis (DIA) is becoming central to the quantitative evaluation of tissue biomarkers for discovery, diagnosis and therapeutic selection for the delivery of precision medicine. In this ...study, automated DIA using a new purpose-built software platform (QuPath) is applied to a cohort of 293 breast cancer patients to score five biomarkers in tissue microarrays (TMAs): ER, PR, HER2, Ki67 and p53. This software is able to measure IHC expression following fully automated tumor recognition in the same immunohistochemical (IHC)-stained tissue section, as part of a rapid workflow to ensure objectivity and accelerate biomarker analysis. The digital scores produced by QuPath were compared with manual scores by a pathologist and shown to have a good level of concordance in all cases (Cohen's κ>0.6), and almost perfect agreement for the clinically relevant biomarkers ER, PR and HER2 (κ>0.86). To assess prognostic value, cutoff thresholds could be applied to both manual and automated scores using the QuPath software, and survival analysis performed for 5-year overall survival. DIA was shown to be capable of replicating the statistically significant stratification of patients achieved using manual scoring across all biomarkers (P<0.01, log-rank test). Furthermore, the image analysis scores were shown to consistently lead to statistical significance across a wide range of potential cutoff thresholds, indicating the robustness of the method, and identify sub-populations of cases exhibiting different expression patterns within the p53 and Ki67 data sets that warrant further investigation. These findings have demonstrated QuPath's suitability for fast, reproducible, high-throughput TMA analysis across a range of important biomarkers. This was achieved using our tumor recognition algorithms for IHC-stained sections, trained interactively without the need for any additional tumor recognition markers, for example, cytokeratin, to obtain greater insight into the relationship between biomarker expression and clinical outcome applicable to a range of cancer types.
Formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide for tissue storage. This method preserves the tissue integrity but causes extensive damage to nucleic acids ...stored within the tissue. As methods for measuring gene expression such as RT-PCR and microarray are adopted into clinical practice there is an increasing necessity to access the wealth of information locked in the Formalin fixation and paraffin embedding archives. This paper reviews the progress in this field and discusses the unique opportunities that exist for the application of these techniques in the development of personalized medicine.
Germline mutations of the BRCA1 gene account for approximately 5% of breast and ovarian cancer cases, and lower than normal BRCA1 expression or function may be an important contributing factor in ...sporadic cancers. The major role of BRCA1 is to respond to DNA damage by participating in cellular pathways for DNA repair, mRNA transcription, cell cycle regulation, and protein ubiquitination. Because most chemotherapeutic agents function by directly or indirectly damaging DNA, the role of BRCA1 as a regulator of chemotherapy-induced DNA damage has been the subject of an increasing number of investigations. We review published preclinical and clinical evidence that the level of BRCA1 function in an individual patient's tumor can guide the choice of chemotherapeutic agents for breast and ovarian cancer. We conclude that a loss of BRCA1 function is associated with sensitivity to DNA-damaging chemotherapy and may also be associated with resistance to spindle poisons. We recommend that prospective clinical studies investigating the role of BRCA1 in the response to chemotherapy be conducted.
Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in ...the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically.
The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.
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